It is well documented that malignant transformation of epithelial cells is associated with structural changes in cell surface carbohydrates. Many clinical studies have correlated those cancer-associated carbohydrate antigens with poor clinical prognosis, including metastasis. Carbohydrate-dependent cancer metastasis has been observed, but the mechanisms underlying this process are not yet defined. We previously identified IELLQAR (l-peptide) that mimics the epitope of anti-Lewis A antibody by screening peptide displaying phage libraries. When l-peptides were injected into wild type mice, they inhibited sLex-dependent melanoma lung colonization. However, lung colonization also occurs in mutant mice lacking both E- and P-selectins and l-peptide inhibits the colonization, excluding the involvement of these selectins in this process. These findings suggest that l-peptide interference with colonization does not require E- and P-selectins. Our preliminary data show that the l-peptide receptors are pre-mRNA splicing factor (Sfrs) and AnnexinAI (Anxal). The PI also studied the in vivo roles of the Golgi processing a-mannosidase II (Mil) and alphamannosidase llx (MX). Mil/MX double null mouse embryos showed no complex type A/-glycans, indicating that Mil and MX together are responsible for the complex type N-glycan synthesis. Mil/MX double nulls were lethal shortly after birth. Based on these findings, the specific aims are: (1) To determine A/-glycan-based L-selectin ligand activity in vivo using conditional Mil/MX double null mice. Endothelial and hematopoietic cell-specific Mil/MX double knockouts (Tie2-Cre:Mllftaxp/floxp/MX-/-) will be generated and analyzed for L-selectin ligand activity in high endothelial venules (HEVs), (2) to define carbohydrate-binding activity of Sfrs and Anxal, (3) to determine if targeted apoptosis occurs by Anxal binding. Anxal has been identified as a tumor-specific endothelial marker, and we identified IFpeptide, which binds preferentially to Anxal. Thus we will develop a method to target apoptosis to endothelial cells of tumor vasculature in the mouse using IF-peptide, and (4) to determine if Anxal expressed on the endothelial surface promotes angiogenesis and identify mechanisms underlying Anxal-dependent endothelial cell activation including binding to heparan sulfate. These studies will provide information for better understand of the mechanisms underlying carbohydrate-dependent cancer metastasis and help for developing new therapeutic strategies against epithelial cancer.